Technical Abstract:
Citrus Huanglongbing (HLB) or yellow shoot disease (ex. greening disease) is highly destructive to citrus production worldwide. Understanding the etiology of HLB is critical for managing the disease. HLB is currently known to be associated with infection by Candidatus Liberibacter asiaticus in China. However, Koch’s postulates have not been fulfilled. In addition, other plant pathogens also may be involved in HLB. In a survey performed in Guangdong Province, P. R. China in 2006 and 2007, 141 citrus samples showing typical symptoms of HLB from 11 different cities were collected. PCR using phytoplasma-specific primer sets fU5/rU3 nested with primer set P1/P7 identified 110 (78.0%) positive samples. A 1,785-bp amplicon was obtained with primer set P1/P7. Analysis showed a 100% identity of this sequence in the region of 16S rDNA and 16S-23S intergenic spacer to three strains of Candidatus Phytoplasma asteri [onion yellows (Japan), aster yellows ‘watercress’ (Hawaii), and valeriana yellows (Lithuania)]. Of the 141 samples, 89 (63.1%) samples were positive for Ca. Liberibacter asiaticus alone. Sixty-nine (48.9%) of the samples was positive for both phytoplasma and Ca. L. asiaticus. Transmission electron microscopy (TEM) showed the presence of both walled and wall-less bodies in symptomatic citrus tissue but at low titer. The presence of phytoplasma with HLB was further demonstrated using the model host periwinkle (Catharanthus roseus (L.) G. Don.). HLB-associated bacteria were transmitted from symptomatic citrus to periwinkle through dodder (Cuscuta campestris Yunck). PCR of infected periwinkle samples detected the same phytoplasma as those in citrus and also detected Ca. L. asiaticus. In addition to yellowing/mottling, infected periwinkle showed symptoms of virescence and phyllody which are associated commonly with phytoplasmal diseases. TEM analysis of affected periwinkle revealed pleomorphic and wall-less organisms, characteristic of phytoplasmas, filling some phloem sieve tubes. This study showed that in addition to Ca. L. asiaticus, a phytoplasma related to Ca. P. asteri is associated with citrus showing HLB symptoms in Guangdong.